Nicotinic acid and picolinic acid derived near-infrared fluorophores

ABSTRACT

This invention relates to new fluorescent chemical entities, including fluorescent molecules that comprise a carboxyl or carbonyl functionalized pyridine moiety. This invention also relates to the corresponding reactive versions of such molecules. This invention also relates to the corresponding conjugates with moieties such as peptides, proteins, various biomolecules, carbocyclic and heterocyclic compounds, sugars, and their uses thereof.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/714,074, filed on Sep. 2, 2005.

The entire teachings of the above application are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Optical imaging is an evolving clinical imaging modality that usespenetrating light rays to create images. Light in the red andnear-infrared (NIR) range (600-1200 nm) is used to maximize tissuepenetration and minimize absorption from natural biological absorberssuch as hemoglobin and water. (Wyatt, Phil. Trans. R. Soc. London B352:701-706, 1997; Tromberg, et al., Phil. Trans. R. Soc. London B352:661-667, 1997).

Besides being non-invasive, optical imaging methods offer a number ofadvantages over other imaging methods: they provide generally highsensitivity, do not require exposure of test subjects or lab personnelto ionizing radiation, can allow for simultaneous use of multiple,distinguishable probes (important in molecular imaging), and offer hightemporal and spatial resolution (important in functional imaging and invivo microscopy, respectively).

In fluorescence imaging, filtered light or a laser with a definedbandwidth is used as a source of excitation light. The excitation lighttravels through body tissues. When it encounters a reporter molecule(i.e., contrast agent or imaging has detectably different propertiesfrom the excitation light. The resulting emitted light then can be usedto construct an image.

Most optical imaging techniques have relied on the use of organic andinorganic fluorescent molecules as the reporter molecule.

Fluorescent dyes are generally known and used for fluorescence labelingand detection of various biological and non-biological materials byprocedures such as fluorescence microscopy, fluorescence immunoassay andflow cytometry. A typical method for labeling such materials withfluorescent dyes is to create a fluorescent complex by means of bondingbetween suitable groups on the dye molecule and compatible groups on thematerial to be labeled. In this way, materials such as cells, tissues,amino acids, proteins, antibodies, drugs, hormones, nucleotides, nucleicacids, lipids and polysaccharides and the like may be chemically labeledand detected or quantified, or may be used as fluorescent probes whichcan bind specifically to target materials and detected by fluorescencedetection methods. Brightly fluorescent dyes permit detection orlocation of the attached materials with great sensitivity.

Certain carbocyanine or polymethine dyes have demonstrated utility aslabeling reagents for a variety of biological applications, e.g. U.S.Pat. No. 4,981,977 to Southwick, et al. (1991); U.S. Pat. No. 5,268,486to Waggoner et al. (1993); U.S. Pat. No. 5,569,587 to Waggoner (1996);U.S. Pat. No. 5,569,766 to Waggoner et al. (1996); U.S. Pat. No.5,486,616 to Waggoner et al. (1996); U.S. Pat. No. 5,627,027 to Waggoner(1997); U.S. Pat. No. 5,808,044 to Brush, et al. (1998); U.S. Pat. No.5,877,310 to Reddington, et al. (1999); U.S. Pat. No.6,002,003 to Shen,et al. (1999); U.S. Pat. No. 6,004,536 to Leung et al. (1999); U.S. Pat.No. 6,008,373 to Waggoner, et al. (1999); U.S. Pat No. 6,043,025 toMinden, et al. (2000); U.S. Pat. No. 6,127,134 to Minden, et al. (2000);U.S. Pat. No. 6,130,094 to Waggoner, et al. (2000); U.S. Pat. No.6,133,445 to Waggoner, et al. (2000); also WO 97/40104, WO 99/51702, WO01/21624, and EP 1 065 250 A1; and Tetrahedron Letters 41, 9185-88(2000); all of the above incorporated by reference.

Comprehensive reviews regarding polymethine dyes have been by written byL. G. S. Brooker, “The Theory of the Photographic Process” Mees Ed.,Macmillan, New York, (1942), p. 987 and (1966), p. 198; Frances M.Hamer, in “The Chemistry of Heterocyclic Compounds”, Vol 18, “TheCyanine Dyes and Related Compounds”, Weissberger, Ed, WileyInterscience, New York, (1964); G. E. Ficken, “The Chemistry ofSynthetic Dyes”, Vol 4, K. Venkataraman Ed., Academic Press, New York,(1971), p. 211; A. I. Kiprianov, Usp. Khim., 29, 1336, (1960), 35, 361(1966), 40, 594 (1971); D. W. Heseltine, “The Theory of the PhotographicProcess”, 4.sup.th edition, James Ed., Macmillan, New York, (1977),chapter 8, “Sensitising and Desensitising Dyes”; S. Daehne, Phot. Sci.Eng., 12, 219 (1979); D. J. Fry, “Rodd's Chemistry of Carbon Compounds”,“Cyanine Dyes and Related Compounds”, Vol. IVb, chapter 15, p. 369Elsevier, Amsterdam, (1977); Supplement to Vol. IVb, 2.sup.nd Edition(1985), p. 267; H. Zollinger, “Color Chemistry”, VCH, Weinheim (1987),chapters 3 and 14; D. M. Stunner, “The Chemistry of HeterocyclicCompounds”, “Special Topics in Heterocyclic Chemistry”, chapter VIII,“Synthesis and Properties of Cyanine and Related Dyes”, Weissberger Ed.,Wiley, New York, (1977); “The Kirk-Othmer Encyclopaedia of ChemicalTechnology” Vol 7, p. 782, “Cyanine Dyes”, Wiley, New-York, (1993).

To be useful as a label, a dye has to be provided with a suitable sidechain containing a functional group. The method and site of introductionof a side chain containing a functional group into the structure for thepurpose of conjugation, or binding to another molecule, represents theinnovative step in the inventions concerning the use of the dye as alabeling reagent. Typically, only one such functionalized side arm isused in order to avoid cross-linking or purification problems. Oneaspect in the design of polymethine labeling reagents has been to attachthe functionalized side arm to one of the heterocyclic nuclei of thedye, formula (1):

Z¹-PML-Z²   (1)

See, for instance: J. S. Lindsey, P. A. Brown, and D. A. Siesel,“Visible Light-Harvesting in Covalently-Linked Porphyrin-Cyanine Dyes,Tetrahedron, 45, 4845, (1989); R. B. Mujumdar, L. A. Ernst, S. R.Mujumdar, and A. S. Waggoner, “Cyanine Dye Labelling Reagents ContainingIsothiocyanate Groups”, Cytometry, 10, 11 (1989); L. A. Ernst, R. K.Gupta, R. B. Mujumdar, and A. S. Waggoner, “Cyanine Dye LabellingReagents for sulphydryl Groups”, Cytometry, 10, 3, (1989); P. L.Southwick P. L., L. A. Ernst, E. W. Tauriello, S. R. Parker, R. B.Mujumdar, S. R. Mujumdar, H. A. Clever, and A. S. Waggoner, “Cyanine DyeLabelling Reagents-Carboxymethylindocyanine Succinimidyl Esters”,Cytometry 11, 418 (1990); R. B. Mujumdar, L. A. Ernst, Swati R.Mujumdar, C. J. Lewis, and A. S. Waggoner, “Cyanine Dye LabellingReagents: Sulfoindocyanine Succinimidyl Esters”, Bioconjugate Chemistry,4, 105, (1993); A. J. G. Mank, E. J. Molenaar, H. Lingeman, C. Goojer,U. A. Th. Brinkman, and N. H. Velthorst, “Visible Diode Laser InducedFluorescence Detection in Liquid Chromatography after PrecolumnDerivatisation of Thiols”, Anal. Chem., 65, 2197, (1993); H. Yu., J.Chao, D. Patek, S. R. Mujumdar, and A. S. Waggoner, “Cyanine dye dUTPanalogs for enzymatic labelling of DNA Probes”, Nucl. Acids Res 22,3226, (1994); Z. Zho, J. Chao, H. Yu, and A. S. Waggoner, “Directlylabelled DNA probes using fluorescent nucleotides with different lengthlinkers”, Nucl. Acids, Res, 22, 3226. A. J. G. Mank, H. T. C. van derLaan, H. Lingeman, Cees Goojer, U. A. Th. Brinkman, and N. H. Velthorst,“Visible Diode Laser-Induced Fluorescence Detection in LiquidChromatography after Precolumn Derivatisation of Amines”, Anal. Chem.,67, 1742, (1995); S. R. Mujumdar, R. B. Mujumdar, C. M. Grant, and A. S.Waggoner, “Cyanine Labelling Reagents: sulfobenzoindocyaninesuccinimidyl esters”, Bioconjugate Chemistry, 7, 356, (1996). PatentLiterature: P. L. Southwick, and A. S. Waggoner, “Intermediate for andFluorescent Cyanine Dyes containing Carboxylic Acid Groups”, U.S. Pat.No. 4,981,977, Jan. 1, 1991; A. S. Waggoner, L. A. Ernst, and Mujumdar,R. B., “Method for Labelling and Detecting Materials EmployingArylsulfonate Cyanine Dyes”, U.S. Pat. No. 5,268,486, Dec. 7, 1993; A.S. Waggoner, “Cyanine Dyes as Labelling Reagents for Detection ofBiological and Other Materials by Luminescence Methods”, U.S. Pat. No.5,627,027, May 6, 1996; A. S. Waggoner, and R. B. Mujumdar, “RigidisedTrimethine Cyanine Dyes”, WO99/311181; G.-Y. Shen, T. S. Dobashi,“Cyanine Dye Activating Group with Improved Coupling Selectivity”; T. S.G. M. Little, R. Raghavachari; N. Narayanan; H. L. Osterman,“Fluorescent Cyanine Dyes”, U.S. Pat. No. 6,027,709, Feb. 22, 2000.

The general synthetic strategy necessary to prepare these labelingreagents is as follows. First, a quaternized nitrogen heterocycle Z¹ isprepared. Then, this heterocyclic base is reacted with a polymethinelinker (PML) that is an electrophilic reagent such asPhNH—(CH═CH)_(n)—CH═NHPh.HCl or RO—(CH═CH)_(n)—CH(OR)₂, where Ph is aphenyl ring and R a methyl or ethyl group, to obtain a so-calledhemicyanine dye, Z¹—(CH═CH)_(n)NHPh or Z¹—(CH═CH)_(n)NAcPh, where Ac isthe acetyl radical, or Z¹—(CH═CH)_(n)—OR. These intermediates are thenreacted with a different quaternary nitrogen heterocycle, Z². Thefunctionalized side arm can be attached either to the first or to thesecond quaternized nitrogen heterocycle.

The hemicyanine intermediates, however, can be difficult to obtain ingood yields and/or in a pure form (see, for example, F. M. Hamer, “SomeUnsymmetrical Pentamethincyanine Dyes and their TetramethinIntermediates”, J. Chem. Soc., 32 (1949) and R. B. Mujumdar, L. A.Ernst, Swati R. Mujumdar, C. J. Lewis, and A. S. Waggoner, “Cyanine DyeLabelling Reagents: Sulfoindocyanine Succinimidyl Esters”, BioconjugateChemistry, 4, 105, (1993).

Thus polymethine dyes that are efficient and easy to produce as well assuitable for preparing conjugates with biomolecules are desirable.

SUMMARY OF THE INVENTION

Fluorescent dye compounds with a polymethine linker bridge that arebright, highly fluorescent dyes that absorb and emit between about 440and about 1100 nm, have now been discovered.

The present invention is directed to fluorescent dye compounds with apolymethine linker bridge that has been modified by a carboxylcontaining pyridine ring which are capable of producing strongfluorescence in the 600 nm to 900 nm region of the spectrum and alsocontain functional groups and/or solubilizing groups which render thedye suitable for covalent labeling, in particular to biologicalmolecules and other target materials.

In one embodiment, the present invention is directed to a polymethinefluorochrome compound represented by formulae (2) or a salt thereof:

X is independently selected from the group consisting ofC(CH₂Y₁)(CH₂Y₂), O, S, and Se;

Y₁ and Y₂ are independently selected from the group consisting of H,C₁-C₂₀ aliphatic group and a C₁-C₂₀ aliphatic group substituted with—OR*, N(R*)₂ or —SR*;

W represents a benzo-condensed, a naphtho-condensed or apyrido-condensed ring;

R₁ is selected from the group consisting of H, (CH₂)_(x)CH₃,(CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein x is an integer selected from0 to 6 and n is an integer selected from 2 to 6;

R₄ is selected from the group consisting of H, (CH₂)_(x)CH₃,(CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein x is an integer selected from0 to 6 and n is an integer selected from 2 to 6;

R₂ and R₃ are independently selected from the group consisting of H,carboxylate, carboxylic acid, carboxylic ester, amine, amide,sulfonamide, hydroxyl, alkoxyl, a sulphonic acid moiety and a sulphonatemoiety;

Q is selected from a group consisting of a heteroaryl ring substitutedwith a carboxyl group or 6-membered heteroaryl ring substituted with acarbonyl group.

In another embodiment present invention is directed to a polymethinefluorochrome compound represented by formulae (2) or a salt thereofwhen:

X is independently selected from the group consisting ofC(CH₂Y₁)(CH₂Y₂), O, S, and Se;

Y₁ and Y₂ are independently selected from the group consisting of H,C₁-C₂₀ aliphatic group and a C₁-C₂₀ aliphatic group substituted with—OR*, N(R*)₂ or —SR*;

W represents a benzo-condensed, a naphtho-condensed or apyrido-condensed ring;

R₁ is selected from the group consisting of (CH₂)_(x)CH₃, (CH₂)_(n)SO₃ ⁻and (CH₂)_(n)SO₃H, wherein x is an integer selected from 0 to 6 and n isan integer selected from 2 to 6;

R₄ is selected from the group consisting of (CH₂)_(x)CH₃, (CH₂)_(n)SO₃ ⁻and (CH₂)_(n)SO₃H, wherein x is an integer selected from 0 to 6 and n isan integer selected from 2 to 6;

R₂ and R₃ are independently selected from the group consisting of H,carboxylate, carboxylic acid, carboxylic ester, amine, amide,sulfonamide, hydroxyl, alkoxyl, a sulphonic acid moiety and a sulphonatemoiety;

Q is selected from a group consisting of a heteroaryl ring substitutedwith a carboxyl group or 6-membered heteroaryl ring substituted with acarbonyl group.

In another embodiment, the present invention is directed to abiocompatible fluorescent molecule comprising one or more biomoleculeschemically linked to a compound of the present invention.

In another embodiment, the present invention is directed to abiocompatible fluorescent molecule comprising a biomolecule chemicallylinked to a compound of the present invention, wherein the compound hasan absorption and emission maxima between about 500 nm and about 900 nm.

In another embodiment, the present invention is directed to abiocompatible fluorescent molecule comprising a biomolecule chemicallylinked to a compound of the present invention wherein the biocompatiblefluorescent molecule is activated after target interaction.

In another embodiment, the present invention is directed to abiocompatible fluorescent molecule comprising a biomolecule chemicallylinked to a compound of the present invention wherein the biocompatiblefluorescent molecule has a high binding affinity to a target.

In another embodiment, the present invention is directed to a method ofin vivo optical imaging, the method comprising:

-   -   (a) administering to a subject a compound or biocompatible        fluorescent molecule of the present invention    -   (b) allowing time for the compound or biocompatible fluorescent        molecule to distribute within the subject or to contact or        interact with a biological target;    -   (c) illuminating the subject with light of a wavelength        absorbable by the compound or biocompatible fluorescent        molecule; and    -   (d) detecting the optical signal emitted by the compound or        biocompatible fluorescent molecule.

In another embodiment, the present invention is directed to abiocompatible fluorescent molecule comprising a biomolecule chemicallylinked to a compound of the present invention, wherein the biocompatiblefluorescent molecule is a labeled cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a fluorescence image of a compound of the present invention intumors of a female NU/NU mice (6-8 weeks old) after 24 hrs on afluorescence reflectance system (FRI, Kodak 2000MM) system.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

The present invention is directed to bright, highly fluorescentcompounds (dyes) that absorb and/or emit between about 440 and about1100 nm, between about 550 and about 800 nm, between about 500 and about900 nm or between about 600 and about 900 nm and conjugates thereof. Itwill be appreciated that compounds (fluorochromes) with excitation andemission wavelengths in other spectrums, such as the visible andultraviolet light spectrum, are also encompassed by the presentinvention.

The structures of the compounds of the present invention, in general arebased on nicotinic acid and picolinic acid derivatives that confer highquantum yields of fluorescence. Moreover, in certain embodiments of thepresent invention the compounds contain functional or reactive groupswhich may be used to chemically link with complementary groups on targetmolecules.

“Chemically linked” or “chemically link” means connected by anattractive force between atoms strong enough to allow the combinedaggregate to function as a unit. This includes, but is not limited to,chemical bonds such as covalent bonds, non-covalent bonds such as ionicbonds, metallic bonds, and bridge bonds, hydrophobic interactions,hydrogen bonds, and van der Waals interactions. This also includescrosslinking or caging.

The term “compounds,” as used herein, refers to “polymethinefluorochromes”, “fluorochromes”, “fluorescent dyes”, “cyanine dyes”“carbocyanine dyes” and “dyes” of the instant invention. These terms areused interchangeably to refer to the compounds of the instant invention.

In one embodiment, the compounds of the invention comprise twoheterocyclic ring systems bound together by a polymethine linker (PML),according to the formula (1):

Z¹-(PML)-Z²   (1)

wherein Z¹ is a heterocyclic ring system such as an indolinium ring, Z²is a second heterocyclic ring system, such as, an indolinium ring, andPML is a polymethine linker that is substituted with a carboxylcontaining heterocyclic ring, such as, pyridine. The Z¹ and Z² ringsystems are optionally further substituted by a variety of substituentsor are fused to additional rings that are optionally furthersubstituted.

In one aspect, the compounds of the present invention are furthersubstituted one or more times by sulfo or sulfoalkyl. By “sulfo” ismeant sulfonic acid, or salt of sulfonic acid (sulfonate). Similarly, by“carboxyl” is meant carboxylic acid, carboxylate ester or salt ofcarboxylic acid. “Phosphate” is an ester of phosphoric acid, andincludes salts of phosphate. “Phosphonate” means phosphonic acid andincludes salts of phosphonate. Similarly for “carbonyl” groups such as,but not limited to carbonyl halode, (e.g., chloride) and carboxamide areincluded. As used herein, unless otherwise specified, the alkyl portionsof substituents such as alkyl, alkoxy, arylalkyl, alkylamino,dialkylamino, trialkylammonium, or perfluoroalkyl are optionallysaturated, unsaturated, linear or branched, and all alkyl, alkoxy,alkylamino, and dialkylamino substituents are themselves optionallyfurther substituted by carboxy, sulfo, amino, or hydroxy.

In one embodiment the present invention is directed to a compoundrepresented by formulae (2) or a salt thereof, wherein:

wherein X is independently selected from C(CH₂Y₁)(CH₂Y₂), O, S, Se; andY₁ and Y₂ are independently selected from H and C₁-C₂₀ alkyl that islinear or branched, saturated or unsaturated, optionally containing N,S, O in various forms; W represents non-metal atoms required to form abenzo-condensed, a naphtho-condensed or a pyrido-condensed ring. R₁ isselected from the group consisting of H, (CH₂)_(n)CH₃, (CH₂)_(n)SO₃ ⁻and (CH₂)_(n)SO₃H, wherein n is an integer selected from 0 to 6 when R₁is (CH₂)_(n)CH₃, and n is an integer selected from 2 to 6 when R₁ is(CH₂)_(n)SO₃ ⁻ or (CH₂)_(n)SO₃H. R₄ is selected from the groupconsisting of H, (CH₂)_(n)CH₃, (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, whereinn is an integer selected from 0 to 6 when R₄ is (CH₂)_(n)CH₃, and n isan integer selected from 2 to 6 when R₄ is (CH₂)_(n)SO₃ ⁻ or(CH₂)_(n)SO₃H. R₂ and R₃ are independently selected from the groupconsisting of H, carboxylate, carboxylic acid, carboxylic ester, amine,amide, sulfonamide, hydroxyl, alkoxyl, a sulphonic acid moiety and asulphonate moiety. Q is selected from a group consisting of a carboxylfunctionalized nitrogen containing heterocyclic ring. Q is selected froma group consisting of a carbonyl functionalized nitrogen containing6-membered heterocyclic ring, such as pyridine. Q is selected from agroup consisting of isonicotinic acid, nicotinic acid and picolinicacid. Q is selected from the groups shown:

Herein, the carboxyl group is also in the form of an ester, an activatedester or carbonyl halide that is capable of reacting with nucleophiles.The carboxyl group is also in a form selected from the groupCO—Obenzotriazolyl, CO—O—N-succinimidyl, CO—Otetrafluorophenyl,CO—Opentafluorophenyl, CO—Oimidazolyl, CO—Op-nitrophenyl.Such molecules of formula (2) are chemically linked to biocompatiblefluorescent molecules for a variety of applications including in vivoimaging.

In another embodiment the present invention is directed to a compoundrepresented by formulae (2) or a salt thereof; wherein:

wherein X is independently selected from C(CH₂Y₁)(CH₂Y₂), O, S, Se; andY₁ and Y₂ are independently selected from H and C₁-C₂₀ alkyl that islinear or branched, saturated or unsaturated, optionally containing N,S, O in various forms; W represents non-metal atoms required to form abenzo-condensed, a naphtho-condensed or a pyrido-condensed ring. R₁ isselected from the group consisting of H, (CH₂)_(n)CH₃, (CH₂)_(n)SO₃ ⁻and (CH₂)_(n)SO₃H, wherein n is an integer selected from 0 to 6 when R₁is (CH₂)_(n)CH₃, and n is an integer selected from 2 to 6 when R₁ is(CH₂)_(n)SO₃ ⁻ or (CH₂)_(n)SO₃H. R₄ is selected from the groupconsisting of (CH₂)_(n)CH₃, (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein nis an integer selected from 0 to 6 when R₄ is (CH₂)_(n)CH₃, and n is aninteger selected from 2 to 6 when R₄ is (CH₂)_(n)SO₃ ⁻ or (CH₂)_(n)SO₃H.R₂ and R₃ are independently selected from the group consisting of H,carboxylate, carboxylic acid, carboxylic ester, amine, amide,sulfonamide, hydroxyl, alkoxyl, a sulphonic acid moiety and a sulphonatemoiety. Q is selected from a group consisting of a carboxylfunctionalized nitrogen containing heterocyclic ring. Q is selected froma group consisting of a carbonyl functionalized nitrogen containing6-membered heterocyclic ring, such as pyridine. Q is selected from agroup consisting of isonicotinic acid, nicotinic acid and picolinicacid. Q is selected from the groups shown:

Herein, the carboxyl group is also in the form of an ester, an activatedester or carbonyl halide that is capable of reacting with nucleophiles.The carboxyl group is also in a form selected from the groupCO—Obenzotriazolyl, CO—O—N-succinimidyl, CO—Otetrafluorophenyl,CO—Opentafluorophenyl, CO—Oimidazolyl, CO—Op-nitrophenyl.Such molecules of formula (2) are chemically linked to biocompatiblefluorescent molecules for a variety of applications including in vivoimaging.

In one embodiment for structural formula (2) Q is selected from a groupconsisting of a heteroaryl ring substituted with a carboxyl or6-membered heteroaryl ring substituted with a carbonyl group.

In certain embodiments:

a) the carboxyl substituent on Q is selected from the group consistingof an ester, an activated ester; or

b) the carboxyl substituent on Q is selected from the group consistingof CO—Obenzotriazolyl, CO—ON-succinimidyl, CO—Otetrafluorophenyl,CO—Opentafluorophenyl, CO—Oimidazolyl, and CO—Op-nitrophenyl; and

c) the carbonyl substituent on Q is in the form of a carbonyl halide.

In another embodiments, Q is a carboxyl substituted nitrogen containingheterocyclic ring.

In another embodiments, Q is selected from the group consisting ofcarboxyl substituted pyridine, pyrimidone, pyrazine, and pyridazine.

In another embodiments, Q is carboxyl substituted pyridine.

In another embodiments, Q is selected from a group consisting ofisonicotinic acid, nicotinic acid and picolinic acid.

In another embodiments, Q is represented by a structural formulaselected from a group consisting of:

In another embodiments, Q is a carbonyl substituted nitrogen containing6-membered heteroaryl ring

In another embodiments, Q is a carbonyl substituted pyridine.

In the immediately preceding nine embodiments, the terms heterocyclicring and heteroaryl ring both refer to heteroaryl ring as definedherein, such as pyridine.

In another embodiment, the present invention provides compoundsrepresenting Z¹-(PML)-Z² by the formula (2):

wherein, W represents non-metal atoms required to form abenzo-condensed, a naphtho-condensed or a pyrido-condensed ring.

In one aspect W represents the atoms necessary to form one to two fusedaromatic rings having 6 atoms in each ring, which atoms are selectedfrom —CH, —C, —CR₇, and —NR₈, where R₈ is 0 or 1 (such that each ringnitrogen is either quaternized or not), and each R₇ independentlycontains sulfo, trifluoromethyl, or halogen; R₈ independently contains aC₁-C₈ alkyl, in turn containing independently an H, amino or sulfo.

Incorporation of one or more non-hydrogen substituents on the fusedrings can be used to tune the absorption and emission spectrum of theresulting dye.

Selected examples of the basic structure of Z¹ and Z² are shown below.These basic structures (3-6) are optionally further substituted asdescribed in this section.

In one embodiment, X is independently selected from O, S, Se,—C(CH₂Y₁)(CH₂Y₂), wherein Y₁ and Y₂ are independently selected from Hand C₁-C₂₀ alkyl that is linear or branched, saturated or unsaturated,optionally containing N, S, O in various forms.

In another embodiment Y₁ and Y₂ together are part of a cyclic ring; or Xis independently selected from —CR₅R₆, wherein R₅ and R₆, which may bethe same or different, are alkyl, cycloalkyl, or arylalkyl, togetherpart of a cyclic system and optionally further substituted.

In one aspect of the invention, R₂ and R₃ taken in combination completea five or six-membered ring.

The substituent R₁ is typically selected from the group consisting of(CH₂)_(n)CH₃, (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein n is an integerselected from 0 to 6 when R₁ is (CH₂)_(n)CH₃, and n is an integerselected from 2 to 6 when R₁ is (CH₂)_(n)SO₃ ⁻ or (CH₂)_(n)SO₃H. In oneaspect, R₁ is (CH₂)₃SO₃ ⁻ or (CH₂)₄SO₃ ⁻ or CH₂CH₃. In one aspect of theinvention R₁ is a substituent containing an aryl sulfonate or an aminogroup or a phthalimido group.

The substituent R₁ is typically selected from the group consisting of H,(CH₂)_(n)CH₃, (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein n is an integerselected from 0 to 6 when R₁ is (CH₂)_(n)CH₃, and n is an integerselected from 2 to 6 when R₁ is (CH₂)_(n)SO₃ ⁻ or (CH₂)_(n)SO₃H. In oneaspect, R₁ is (CH₂)₃SO₃ ⁻ or (CH₂)₄SO₃ ⁻ or CH₂CH₃. In one aspect of theinvention R₁ is a substituent containing an aryl sulfonate or an aminogroup or a phthalimido group.

The substituent R₄ is typically selected from the group consisting of(CH₂)_(n)CH₃, (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein n is an integerselected from 0 to 6 when R₄ is (CH₂)_(n)CH₃, and n is an integerselected from 2 to 6 when R₄ is (CH₂)_(n)SO₃ ⁻ or (CH₂)_(n)SO₃H. In oneaspect, R₄ is (CH₂)₃SO₃ ⁻ or (CH₂)₄SO₃ ⁻ or CH₂CH₃. In one aspect of theinvention R₄ is a substituent containing an aryl sulfonate or an aminogroup or a phthalimido group.

The substituent R₄ is typically selected from the group consisting of H,(CH₂)_(n)CH₃, (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein n is an integerselected from 0 to 6 when R₄ is (CH₂)_(n)CH₃, and n is an integerselected from 2 to 6 when R₄ is (CH₂)_(n)SO₃ ⁻ or (CH₂)_(n)SO₃H. In oneaspect, R₄ is (CH₂)₃SO₃ ⁻ or (CH₂)₄SO₃ ⁻ or CH₂CH₃. In one aspect of theinvention R₄ is a substituent containing an aryl sulfonate or an aminogroup or a phthalimido group.

The substituents R₂ and R₃ are independently selected from the group H,halogens, carboxylate, carboxylic acid, carboxylic esters, amino, amide,alkyl or aryl sulfonamide, hydroxy, alkoxy, aryloxy, sulfate, cyano,nitro, azido, alkylamino, dialkylamino, trialkylammonium, phosphate,phosphate ester, phosphonate, sulphonic acid and a sulphonate moiety. Incertain embodiments, R₂ and R₃ are independently, sulphonic acid or asalt thereof.

In one aspect of the invention, R₂ and R₃ could imply per-substitution,as in per-fluorinated W. Per-fluorination or poly-fluorination can leadto enhancement of fluorescence quantum yield.

As used herein “enhancement” means an increase in the fluorescencequantum yield, by about 5%, about 10%, about 15%, about 25%, about 50%,about 80%, about 90% about 95% about 98% about 99% about 100%.

In one aspect of the invention, the formula Z¹-(PML)-Z² is representedaccording to the formula (7):

In one aspect of the invention, the compounds of the invention aresulfonated one or more times. If the compound of the invention issubstituted by sulfo, it is typically sulfonated at R₂ or R₃ or both,(that is, for example, R₂ and/or R₃ are a sulfonic acid moiety,sulfonate moiety or sulfonamamide) or sulfoalkylated at R₁ or R₄ or both(that is, for example, R₁ and/or R₄ are (CH₂)_(n)SO₃ ⁻ or(CH₂)_(n)SO₃H), or is both sulfonated and sulfoalkylated. In aparticular embodiment, the compounds are sulfonated up to three times(at positions corresponding to R₂ and R₃, and as sulfoalkyl at one of R₁or R₄), leaving one of R₁ or R₄ for the location of a reactive group.

As used herein the terms sulfonic acid and (CH₂)_(n)SO₃H or a sulfonategroup and (CH₂)_(n)SO₃ ⁻ can be used interchangeably. However in certainembodiments the terms a sulfonic acid moiety, sulfonate moiety orsulfonamamide refer to substituents which are attached to the remainderof the molecule by the a sulfonic acid moiety, sulfonate moiety orsulfonamamide moiety, ie., —SO2NR′R″

In certain embodiments, at least one of R₁ to R₃ is or contains asulphonic acid moiety or a sulphonate moiety. In certain embodiments, atleast one of R₁ to R₃ is a sulphonic acid moiety or a sulphonate moiety.

In certain embodiments, R₁ and R₄ are independently (CH₂)_(n)SO₃ ⁻ or(CH₂)_(n)SO₃H.

In one embodiment of the present invention the compounds are sulfonatedup to four times (at R₂ and R₃, and as sulfoalkyl at R₁ and R₄). Inanother embodiment of the present invention the compounds are sulfonatedat least four times (at R₂ and R₃, and as sulfoalkyl at R₁ and R₄). Inanother embodiment of the present invention, any reactive group (orchemically linked molecule) can be attached at Q, in addition thecompounds of the present invention being sulfonated up to four times (atR₂ and R₃, and as sulfoalkyl at R₁ and R₄). In another embodiment of thepresent invention, any reactive group (or chemically linked molecule)can be attached at Q, in addition the compounds of the present inventionbeing sulfonated at least four times (at R₂ and R₃, and as sulfoalkyl atR₁ and R₄). This extra sulfonation, as well as the change in attachmentsite, results in reactive dyes and dye conjugates that are brighter,more soluble in aqueous solutions, and more resistant to thefluorescence quenching that results from dye-dye stacking interactions.

In one aspect of the invention, the formula Z¹-(PML)-Z² is representedaccording to the formula (8):

In one aspect of the invention, the compounds of the invention that aresulfonated one or more times are typically sulfonated at R₂ or R₃, orsulfoalkylated at R₁ or R₄ or both, or is both sulfonated andsulfoalkylated. In particular, the compounds of the present inventionare sulfonated up to four times leaving one of R₁ or R₄ for the locationof a reactive group.

In one embodiment of the present invention the compounds are sulfonatedup to six times (at R₂ and R₃, and as sulfoalkyl at R₁ and R₄). Inanother embodiment of the present invention the compounds are sulfonatedat least six times (at R₂ and R₃, and as sulfoalkyl at R₁ and R₄). Inanother embodiment of the present invention, any reactive group (orchemically linked molecule) can be attached at Q, in addition thecompounds of the present invention being sulfonated up to six times (atR₂ and R₃, and as sulfoalkyl at R₁ and R₄). In another embodiment of thepresent invention, any reactive group (or chemically linked molecule)can be attached at Q, in addition the compounds of the present inventionbeing sulfonated at least six times (at R₂ and R₃, and as sulfoalkyl atR₁ and R₄). This extra sulfonation, as well as the change in attachmentsite, results in reactive dyes and dye conjugates that are brighter,more soluble in aqueous solutions, and more resistant to thefluorescence quenching that results from dye-dye stacking interactions.

In one embodiment, the PML moiety has the formula (9):

wherein Q is selected from a group consisting of carboxyl functionalizedheterocyclic ring (heteroaryl substituted with a carboxy group). In thisembodiment, heterocyclic ring and heteroaryl ring refers to heteroarylring as defined herein, such as pyridine and functionalized refers tosubstituted.

Suitable examples of appropriate PML moieties for compounds of thepresent invention (carbocyanine dyes) have been described in theliterature, including PML moieties that incorporate nonhydrogensubstituents, ring structures, and rigidizing elements (U.S. Pat. No.5,831,098 to Ollmann, Jr (1998); U.S. Pat. No. 6,086,737 to Patonay etal. (2000); U.S. Pat. No. 6,048,982 to Waggoner (2000); and U.S. Pat.No. 5,453,505 to Lee et al. (1995); U.S. Pat. No. 5,639,874 toMiddendorf et al. (1997); U.S. Pat. No. 3,864,644 to Lincoln et al(1975); U.S. Pat. No. 4,011,086 to Simson (1977); U.S. Pat. No.6,747,159 to Caputo (2004); all incorporated herein by reference intheir entirety).

In one embodiment the invention is directed to a PML of the formula (9),wherein Q is selected from a group consisting of a functionalizednitrogen-containing heterocyclic ring. One aspect of the invention is aPML of the formula (9), wherein Q is selected from a group consisting ofa substituted nitrogen-containing heteroaryl ring. In these embodiments,heterocyclic ring and heteroaryl ring refers to heteroaryl ring asdefined herein, such as pyridine, and functionalized refers tosubstituted.

In one embodiment of the invention, Q contains at least one substituentwhich is a reactive group that is attached to the heterocyclic(heteroaryl) ring (Q) by a covalent linkage. In one embodiment thecompounds of the present invention which contain a reactive group labela wide variety of organic or inorganic substances that contain or aremodified to contain functional groups with suitable reactivity,resulting in chemical attachment of the compounds of the presentinvention to form a conjugated substance.

As used herein, “reactive group” means a moiety on a compound of thepresent invention or that can be added to a compound of the presentinvention that is capable of chemically reacting with a functional groupon a different compound to form a covalent linkage, or a moiety on adifferent compound that is capable of chemically reacting with afunctional group on compound of the present invention to form a covalentlinkage. Typically the reactive group is an electrophile or nucleophilethat can form a covalent linkage through exposure to the correspondingfunctional group that is a nucleophile or electrophile, respectively.Alternatively, the reactive group is a photoactivatable group, andbecomes chemically reactive only after illumination with light of anappropriate wavelength. Typically, the conjugation reaction between thecompound of the present invention and the substance to be conjugatedresults in one or more atoms of the reactive group to be incorporatedinto a new linkage attaching the dye to the conjugated substance.

One aspect of the invention is a PML of the formula (9), wherein Q isselected from a group consisting of carboxyl functionalized nitrogencontaining heterocyclic ring (heteroaryl substituted with carboxyl). Inthis embodiment, heterocyclic ring and heteroaryl ring refers toheteroaryl ring as defined herein, such as pyridine and functionalizedrefers to substituted.

In one aspect of the invention PML is of the formula (9), wherein Q isselected from a group consisting of carboxyl functionalized nitrogencontaining 6-membered heterocyclic ring (heteroaryl substituted withcarboxyl), such as pyridine, pyrimidone, pyrazine, and pyridazine.(heteroaryl substituted with carboxyl). In this embodiment, heterocyclicring and heteroaryl ring refers to heteroaryl ring as defined herein,such as pyridine and functionalized refers to substituted.

Another aspect of the invention is a PML of the formula (9), wherein Qis selected from a group consisting of carboxyl functionalized nitrogencontaining 6-membered heterocyclic ring, (heteroaryl substituted withcarboxyl), such as pyridine. (heteroaryl substituted with carboxyl). Inthis embodiment, heterocyclic ring and heteroaryl ring refers toheteroaryl ring as defined herein, such as pyridine and functionalizedrefers to substituted.

In another aspect of the invention PML is of the formula (9), wherein Qis selected from a group consisting of nicotinic acid and picolinic acidor a salt thereof.

In one aspect of the invention is a PML of the formula (9), wherein Q isselected from the groups shown:

or a salt thereof.

In another aspect of the invention is a PML of the formula (9), whereinQ is selected from the groups shown:

wherein, the carboxyl group is also in the form of an activated ester(R₁₅) or carbonyl halide (R₁₆═F, Cl, Br) that is capable of reactingwith nucleophiles. The carboxyl group CO—OR₁₅ is also in a form selectedfrom the group CO—Obenzotriazolyl, CO—ON-hydroxysuccinimidyl,CO—Otetrafluorophenyl, CO—Opentafluorophenyl, CO—Oimidazolyl,CO—Op-nitrophenyl.

The PML moiety typically originates from the coupling agent used in thesynthesis of a compound of the present invention. For example,N,N′-diphenylformamidine and triethylorthoformate yields PML moieties.Malonaldehyde bis(phenylimine) hydrochloride, 1,1,3-trimethoxypropane,and 1,1,3,3-tetramethoxypropane and glutaconaldehyde dianil monochloridealso yield PML moieties (dyes).

In one aspect of the invention, the PML moiety is introduced into thedye using the malonodialdehydes moieties shown below:

Accordingly, the present invention provides compounds representingZ¹-(PML)-Z² by the formula (2):

wherein R₁ is (CH₂)_(n)SO₃ ⁻ or (CH₂)_(n)SO₃H.

In one aspect the present invention, is directed to compounds having anyof the formulae (10-11):

In another aspect the present invention is directed to compounds havingany of the formulae (12-15):

In one aspect of the invention when Q is or contains an activated ester,the compound can be chemically linked to bifunctional linkers such asaminoethylmaleimide, aminopropylmaleimide, aminopropylazide,aminopropylthiol, mercaptoethylamine, propargylamine 3-aminopropanol,diaminopropane, and diaminobutane to provide additional reactivefunctional groups in a suitable solvent under neutral or basicconditions.

In one aspect of the invention when Q is or contains NH₂, the compoundof the invention can be chemically linked to bifunctional linkers suchas propargylic acid, succinimidylpyridinedithiopropionate,maleimide-PEG-N-hydroxysuccinimide ester to provide additional reactivefunctional groups in a suitable solvent under neutral or basicconditions.

When a compound of the invention is depicted herein by structureindicating the positions of the double bonds in the rings an polymethinelinker, it is to be understood that the structure also encompasses anyresonance structures as shown, for example, in the figure below:

In one embodiment, the present invention provides compounds representingZ¹-(PML-BM)-Z² by the formula (16):

wherein BM a biomolecule chemically linked via Q residing on the PML.

In one aspect BM is a moiety that changes or alters or enhancesaccumulation, biodistribution, elimination, targeting, binding, and/orrecognition of the compounds (fluorochromes) of the general structureZ¹-(PML)-Z². BMs include but are not limited to antibodies and fragmentsthereof, proteins, peptides, amino acids, antibodies (or antigen-bindingantibody fragments, such as single chain antibodies), glycoproteins,ligands for cell receptors, polysaccharides, nucleosides, aptamers, cellreceptors themselves, enzyme substrates, enzyme cofactors, biotin,hormones, neurohormones, neurotransmitters, growth factors, cytokines,lymphokines, lectins, selectins, toxins, and carbohydrates. Othertargeting and delivery approaches using various biomolecules can also beused, such as folate-mediated targeting (Leamon & Low, Drug DiscoveryToday, 6:44-51, 2001), transferrin, vitamins, carbohydrates and ligandsthat target internalizing receptors, including, but not limited to,asialoglycoprotein receptor, somatostatin, nerve growth factor,oxytocin, bombesin, calcitonin, arginine vasopressin, angiotensin II,atrial natriuretic peptide, insulin, glucagons, prolactin, gonadotropin,various opioids and urokinase-type plasminogen activator. Also includedare membrane, transmembrane, and nuclear translocation signal sequences,which can be derived from a number of sources including, withoutlimitation, viruses and bacteria. BM can also be an organic molecule,polymer, dendrimer, drug, lipid, lipid assembly, therapeutic drugmolecules, polymeric microparticle, cells, or a nanoparticle. In certainembodiments, BMs can also include small molecule drugs, phototherapeuticmolecules and derivatives thereof.

In certain embodiments of the present invention, when BM is chemicallylinked to a compound of the present invention the fluorescence of thecompound of the present invention is enhanced. In certain embodimentsthe fluorescence is enhanced by about 10% , about 25%, about 50% or morethan about 50% which compared with the unlinked compound.

In one aspect of the invention several copies of BM are chemicallylinked to Q via multivalent linkers or linkers containing severalreactive functional groups to form a biocompatible fluorescent moleculeof the structure (Z¹-(PML)-Z²)-((L)_(w)-(BM)q)t, wherein L is a linkeror multivalent linker, and t=1-6, w=1-500 and q=1-500. (L)_(w)represents copies of the same linker or a combination of differentlinkers. Suitable linkers include but are not limited to aminocaproicacid, glutamic acid, and poly-glutamic acid.

In another aspect of the invention, more than one compound(fluorochrome) of the structure Z¹-(PML)-Z² can be chemically linked toa single BM to form a biocompatible fluorescent molecule of thestructure [Z¹-(PML)-Z²]_(k)-BM, wherein k=1-500.

In one embodiment Z¹-(PML-BM)-Z² is a biocompatible fluorescent moleculewherein the compound has an absorption and emission maxima between about440 and about 1100 nm, between about 550 and about 800 nm, between about500 and about 900 nm or between about 600 and about 900 nm.

In one embodiment the compounds (fluorochromes) and biocompatiblefluorescent molecules of the present invention are activated aftertarget interaction. “Activated after target interaction” is meant achange that alters a detectable property, e.g., an optical property, ofthe fluorochromes or biocompatible fluorescent molecules. This includes,but is not limited to, a modification, alteration, or binding (covalentor non-covalent) that results in a detectable difference in properties,e.g., optical properties of the fluorochromes or biocompatiblefluorescent molecules, e.g., changes in the fluorescence signalamplitude (e.g., dequenching and quenching), change in wavelength,fluorescence lifetime, spectral properties, or polarity. In anotherembodiment, a quencher molecule is used to quench the fluorescent signalof the biocompatible fluorescent molecules. By adopting these activatedand unactivated states, it is therefore possible to determine whetherthe fluorochrome or biocompatible fluorescent molecule is active orinactive in a subject by identifying a change in the signal intensity.In addition, the fluorochromes and biocompatible fluorescent moleculescan be designed such that the they exhibit little or no signal untilactivated. Activation can be, without limitation, by enzymatic cleavage,enzymatic conversion, phosphorylation or dephosphorylation, conformationchange due to binding, enzyme-mediated splicing, enzyme-mediatedtransfer, hybridization of complementary DNA or RNA, analyte binding,such as association with an analyte such as Na⁺, K⁺, Ca²⁺, Cl⁻, oranother analyte, change in hydophobicity of the environment and chemicalmodification.

In one embodiment the compounds (fluorochromes) and biocompatiblefluorescent molecules of the present invention have a high bindingaffinity to a target.

Also provided herein is a method of in vivo optical imaging, the methodcomprising (a) administering to a subject a compound (fluorochrome) orbiocompatible fluorescent molecule of the present invention; (b)allowing time for the compound (fluorochrome) or biocompatiblefluorescent molecule to distribute within the subject or to contact orinteract with a biological target; (c) illuminating the subject withlight of a wavelength absorbable by the compound (fluorochrome) orbiocompatible fluorescent molecule; and (d) detecting the optical signalemitted by the compound (fluorochrome) or biocompatible fluorescentmolecule.

The optical signal generated by the compounds (fluorochromes) orbiocompatible fluorescent molecules of the present invention, whethercollected by tomographic, reflectance, planar, endoscopic, microscopic,surgical goggles, video imaging technologies, or other methods such asmicroscopy including intravital and two-photon microscopy, and whetherused quantitatively or qualitatively, is also considered to be an aspectof the invention.

One aspect of the invention is a method wherein the presence, absence,distribution, or level of optical signal emitted by the compound(fluorochrome) or biocompatible fluorescent molecule is indicative of adisease state.

The invention also features a method of using the compounds(fluorochromes) and biocompatible fluorescent molecules of the presentinvention to detect an abnormality in a patient or subject, e.g., anyabnormality associated with a disease such as cancer, a cardiovasculardisease, AIDS, a neurodegenerative disease, an inflammatory disease, arespiratory disease, a metabolic disease, a bone disease or animmunologic disease. The invention also features a method of assessingthe effect of a compound or therapy on a specified molecular target byusing the compounds (compositions) of the present invention, wherein thesubject is imaged prior to and after treatment with the compound ortherapy, and the corresponding images are compared.

The imaging method steps of the present invention can also be repeatedat predetermined intervals thereby allowing for the evaluation ofemitted signal of the Z¹-(PML)-Z² containing compound in a subject orsample over time. The emitted signal may take the form of an image. Thesubject may be a vertebrate animal, for example, a mammal, including ahuman. The animal may also be non-vertebrate, (e.g., C. elegans,drosophila, or other model research organisms, etc.). The sample caninclude, without limitation, cells, cell culture, tissue sections,organs, organ sections, cytospin samples, or the like.

The invention also features an in vivo method for selectively detectingand imaging two or more Z¹-(PML)-Z² containing compounds simultaneously.The method comprises administering to a subject two or more Z¹-(PML)-Z²containing compounds, either at the same time or sequentially, whoseoptical properties are distinguishable. The method therefore allows therecording of multiple events or targets.

The invention also features an in vivo method for selectively detectingand imaging one or more Z¹-(PML)-Z² containing compounds, simultaneouslywith one or more targeted or activatable optical imaging probes, or in adual imaging protocol with magnetic resonance imaging, computedtomography (CT), X-ray, ultrasound, or nuclear medicine imagingmodalities and their respective imaging agents. The method comprisesadministering to a subject one or more imaging probes, either at thesame time or sequentially, including at least one Z¹-(PML)-Z² containingcompound, whose properties are distinguishable from that of the others.In one aspect a dual imaging protocol is optical and magnetic resonanceimaging using Z¹-(PML)-Z² containing compounds sequentially or nearlysimultaneously with magnetic resonance imaging agents, (for example,iron oxide based agents or gadolinium based agents such asgadopentetate). The method therefore, allows the recording of multipleevents or targets using more than one imaging modality or imaging agent.

In another aspect, the invention features an in vitro optical imagingmethod comprising contacting the sample with Z¹-(PML)-Z² containingcompounds; allowing time for the probes to become activated or bind to atarget of interest in the sample; optionally, removing the unboundprobes; illuminating the target with light of a wavelength absorbable bythe Z¹-(PML)-Z² containing compounds; and detecting the optical signalemitted by the Z¹-(PML)-Z² containing compounds.

After administration, detection can occur, for example, by in vitromethods, i.e., flow cytometry or by in vivo imaging methods, i.e.,tomographic, catheter, planar/reflectance systems or endoscopic systems.

In one embodiment, the Z¹-(PML)-Z² containing compounds derived thereof)can be used to label a sample ex vivo. The sample, e.g., cells, can bederived directly from a subject or from another source (e,g., fromanother subject, cell culture etc.). The Z¹-(PML)-Z² containing compoundcan be mixed with the cells to effectively label the cells (covalentlyor non-covalently) and the resulting labeled cells injected into asubject. This method can be used for monitoring trafficking andlocalization of certain cell types, including T-cells, tumor cells,immune cells, stem cells, and other cell types. In particular, thismethod may be used to monitor cell based therapies. The sample can alsobe derived from non-mammalian sources including but not limited toplants, insects, viruses, bacteria, and bacteriophage.

Another aspect of the invention features Z¹-(PML)-Z² containingcompounds that can be used for in vivo imaging and labeling samples exvivo, including cells, without the use of dimethylsulfoxide (DMSO) orother organic solvents (i.e. physiologic buffers or solutions) that aregenerally toxic to biological subjects or samples.

For labeling of BMs or cells, the compounds (fluorochromes) of thepresent invention can be incubated with BMs at various concentrationsfor about 5 minutes to 24 hours or more at about 4°-37° C. After theincubation the free or fluorochrome that has not been chemically linkedto the BM can be removed, such as by chromatography or ultrafiltrationmethods that are well known in the art. For cells, after the incubation,the cells can be centrifuged to create a cell pellet from which thesupernatant is removed. Cells can be resuspended in culture media orphysiologic saline to wash away residual, unbound or free fluorochrome.This can be repeated several times. In this manner, cells can be labeledeither by direct conjugation to internal or external cellular moleculesor by non-specific cell uptake into various intracellular compartments,including but not limited to cytosol, endosomes, nucleus, golgiapparatus, and other intracellular organelles.

Another aspect of the invention features Z¹-(PML)-Z² containing compoundcontaining imaging probes formulated in a pharmaceutical compositionsuitable for administration to animal, including human, subjects. Thepharmaceutical composition can include the nanoparticles and one or morestabilizers in a physiologically acceptable (relevant) carrier.

Another aspect of the invention features biocompatible fluorescentZ¹-(PML)-Z² containing compounds formulated in pharmaceuticalcompositions Suitable for administration to animal, including human,subjects and cells. The pharmaceutical composition can include one ormore stabilizers in a physiologically acceptable (relevant) carrier.

Suitable examples of stabilizers for use in the methods of the presentinvention, include but are not limited to, low molecular weightcarbohydrate, in one aspect it is a linear polyalcohol, such assorbitol, and glycerol; or mannitol. Other low molecular weightcarbohydrates, such as inositol, may also be used. Physiologicallyrelevant carriers can include water, saline, and may further includeagents such as buffers, and other agents such as preservatives that arecompatible for use in pharmaceutical formulations.

The invention also features a method of gene sequence recognition usingfluorescent Z¹-(PML)-Z² containing compounds, labeled nucleic acidrecognition molecules, including DNA, RNA, modified nucleic acid, PNA,molecular beacons, aptamers, or other nucleic acid binding molecules(for example, small interfering RNA or siRNA). The method includes theuse of one or more fluorescent Z¹-(PML)-Z² containing compounds,together with techniques such as hybridization, ligation, cleavage,recombination, synthesis, sequencing, mutation detection, real-timepolymerase chain reactions, in situ hybridization, and the use ofmicroarrays. For example, for detecting a single stranded nucleic acid(i.e., mRNA, cDNA or denatured double-stranded DNA) in a sample, vianucleic acid hybridization principles, a fluorescent Z¹-(PML)-Z²containing compound chemically linked to a single-stranded nucleic acidis contacted with a sample containing one or more single strandednucleic acids and the fluorescence of the fluorescent Z¹-(PML)-Z²containing compound is detected, wherein the presence or level offluorescence signal emitted by the fluorescent Z¹-(PML)-Z² containingcompound indicates the presence or amount of nucleic acid in the sample.

The optical signal generated by the Z¹-(PML)-Z² containing compounds orderivatives thereof, whether collected by tomographic, reflectance,planar, endoscopic, microscopic, surgical goggles or imager, videoimaging technologies, or other methods such as microscopy includingintravital and two-photon microscopy, and whether used quantitatively orqualitatively, is also considered to be an aspect of the invention.

Another aspect of the invention features a kit, which includes theZ¹-(PML)-Z² containing compounds, and optionally, and instructions forusing the fluorochromes or imaging probes for in vivo or in vitroimaging methods. The kit optionally may include components that aid inthe use of the fluorochromes or imaging probes for the disclosedmethods, such as buffers, and other formulating agents; alternatively,the kit may include medical devices that aid in the administration ofthe imaging probes to subjects.

The Z¹-(PML)-Z² containing compounds, and pharmaceutical compositions ofthe present invention can be administered orally, parentally, byinhalation, topically, rectally, nasally, buccally, vaginally, or via animplanted reservoir. The term “parental administration” includesintravenous, intramuscular, subcutaneous, intraarterial, intraarticular,intrasynovial, intrasternal, intrathecal, intraperitoneal,intracisternal, intrahepatic, intralesional, intracranial andintralymphatic injection or infusion techniques. The Z¹-(PML)-Z²containing compound can also be administered via catheters or through aneedle to a tissue.

In one embodiment, an effective amount (which is an amount effective tocause or increase fluorescence) of the compounds of the presentinvention are administered. In one embodiment, between about 1 ng/kg andabout 100 mg/kg, between about 100 ng/kg and 10 mg/kg, between about 1μg/kg and about 5 mg/kg, between about 10 μg/kg and about 2 mg/kg,between about 50 μg/kg and about 1 mg/kg of the compound of the presentinvention is administered.

Preferred Z¹-(PML)-Z² containing compounds have the followingproperties: (1) high quantum yield (e.g., quantum yield greater than 5%in aqueous medium), (2) narrow emission spectrum (e.g., less than 75 nm;more preferably less than 50 nm), (3) spectrally separated absorptionand emission spectra (e.g., separated by more than 20 nm; morepreferably by more than 50 nm), (3) have high chemical stability andphotostability (e.g., retain fluorescent properties after exposure tolight), (4) are non toxic or minimally toxic to cells or subjects atdoses used for imaging protocols, (as measured for example, by LD₅₀ orirritation studies, or other similar methods known in the art) and/or(5) have commercial viability and scalable production for largequantities (e.g., gram and kilogram quantities).

The compounds of the present invention can have one or more sufficientlyacidic proton that can react with a suitable organic or inorganic baseto form a base addition salt. Base addition salts include those derivedfrom inorganic bases, such as ammonium or alkali or alkaline earth metalhydroxides, carbonates, bicarbonates, and the like, and organic basessuch as alkoxides, alkyl amides, alkyl and aryl amines, and the like.Such bases useful in preparing the salts of this invention thus includesodium hydroxide, potassium hydroxide, ammonium hydroxide, potassiumcarbonate, and the like.

The compounds of the present invention having a sufficiently basicgroup, such as an amine can react with an organic or inorganic acid toform an acid addition salt. Acids commonly employed to form acidaddition salts from compounds with basic groups are inorganic acids suchas hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

The term “alkyl” as used herein means a saturated straight-chain,branched or cyclic hydrocarbon. When straight-chained or branched, analkyl group is typically C1-C20, more typically C1-C10; when cyclic, analkyl group is typically C3-C12, more typically C3-C7. Examples of alkylgroups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyland tert-butyl and 1,1-dimethylhexyl.

An “aliphatic group” is non-aromatic, and may optionally contain one ormore units of unsaturation, e.g., double and/or triple bonds. Analiphatic group may be straight-chained or branched and typicallycontains between 1 and 12 carbon atoms, more typically between 1 and 6carbon atoms, and even more typically between 1 and 4 carbon atoms. Oneor more methylene group in an aliphatic group can optionally be replacedby O, S, or NH.

As used herein the term non-aromatic carbocyclic ring or non-aromaticheterocyclic ring as used alone or as part of a larger moiety refers toa non-aromatic carbon or heteroatom containing ring which can besaturated or contain one or more units of unsaturation, having three tofourteen atoms including monocyclic and polycyclic rings in which thecarbocyclic or heterocyclic ring can be fused to one or morenon-aromatic carbocyclic or heterocyclic rings or one or more aromatic(carbocyclic or heterocyclic) rings.

The term “alkoxy” as used herein is represented by —OR**, wherein R** isan alkyl group as defined above.

The term “carbonyl” as used herein is represented by —C(═O)R**, whereinR** is an alkyl group as defined above.

The term “aromatic group” includes carbocyclic aromatic rings andheteroaryl rings. The term “aromatic group” may be used interchangeablywith the terms “aryl”, “aryl ring” “aromatic ring”, “aryl group” and“aromatic group”.

Carbocyclic aromatic ring groups have only carbon ring atoms (typicallysix to fourteen) and include monocyclic aromatic rings such as phenyland fused polycyclic aromatic ring systems in which a carbocyclicaromatic ring is fused to one or more aromatic rings (carbocyclicaromatic or heteroaromatic). Examples include 1-naphthyl, 2-naphthyl,1-anthracyl and 2-anthracyl. Also included within the scope of the term“carbocyclic aromatic ring”, as it is used herein, is a group in whichan aromatic ring is fused to one or more non-aromatic rings (carbocyclicor heterocyclic), such as in an indanyl, phthalimidyl, naphthimidyl,phenanthridinyl, or tetrahydronaphthyl.

The term “heteroaryl”, “heteroaromatic”, “heteroaryl ring”, “heteroarylgroup” and “hetero aromatic group”, used alone or as part of a largermoiety as in “heteroaralkyl” refers to heteroaromatic ring groups havingfive to fourteen members, including monocyclic heteroaromatic rings andpolycyclic aromatic rings in which a monocyclic aromatic ring is fusedto one or more other aromatic ring (carbocyclic or heterocyclic).Heteroaryl groups have one or more ring heteroatoms. Examples ofheteroaryl groups include 2-furanyl, 3-furanyl, N-imidazolyl,2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, oxadiazolyl, oxadiazolyl, 2-oxazolyl, 4-oxazolyl,5-oxazolyl, N-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl,N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl,4-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, triazolyl,tetrazolyl, 2-thienyl, 3-thienyl, carbazolyl, benzothienyl,benzofuranyl, indolyl, quinolinyl, benzothiazole, benzooxazole,benzimidazolyl, isoquinolinyl and isoindolyl. Also included within thescope of the term “heteroaryl”, as it is used herein, is a group inwhich an aromatic ring is fused to one or more non-aromatic rings(carbocyclic or heterocyclic).

The term non-aromatic heterocyclic group used alone or as part of alarger moiety refers to non-aromatic heterocyclic ring groups havingthree to fourteen members, including monocyclic heterocyclic rings andpolycyclic rings in which a monocyclic ring is fused to one or moreother non-aromatic carbocyclic or heterocyclic ring or aromatic ring(carbocyclic or heterocyclic). Heterocyclic groups have one or more ringheteroatoms, and can be saturated or contain one or more units ofunsaturation. Examples of heterocyclic groups include piperidinyl,piperizinyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl,tetrahydroquinolinyl, inodolinyl, isoindolinyl, tetrahydrofuranyl,oxazolidinyl, thiazolidinyl, dioxolanyl, dithiolanyl, tetrahydropyranyl,dihydropyranyl, azepanyl and azetidinyl

The term “heteroatom” means nitrogen, oxygen, or sulfur and includes anyoxidized form of nitrogen and sulfur, and the quaternized form of anybasic nitrogen. Also the term “nitrogen” includes a substitutablenitrogen of a heteroaryl or non-aromatic heterocyclic group. As anexample, in a saturated or partially unsaturated ring having 0-3heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen maybe N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR″ (asin N-substituted pyrrolidinyl), wherein R″ is a suitable substituent forthe nitrogen atom in the ring of a non-aromatic nitrogen-containingheterocyclic group, as defined below. Preferably the nitrogen isunsubstituted.

A substituted aryl group as defined herein contains one or moresubstitutable ring atoms, such as carbon or nitrogen ring atoms.Examples of suitable substituents on a substitutable ring carbon atom ofan aryl or aliphatic group include halogen (e.g., —Br, Cl, I and F),—OH, C1-C4 alkyl, C1-C4 haloalkyl, —NO₂, C1-C4 alkoxy, C1-C4 haloalkoxy,—CN, —NH₂, C1-C4 alkylamino, C1-C4 dialkylamino, —C(O)NH₂, —C(O)NH(C1-C4alkyl), —C(O)(C1-C4 alkyl), —OC(O)(C1-C4 alkyl), —OC(O)(aryl),—OC(O)(substituted aryl), —OC(O)(aralkyl), —OC(O)(substituted aralkyl),—NHC(O)H, —NHC(O)(C1-C4 alkyl), —C(O)N(C1-C4 alkyl)₂, —NHC(O)O—(C1-C4alkyl), —C(O)OH, —C(O)O—(C1-C4 alkyl), —NHC(O)NH₂, —NHC(O)NH(C1-C4alkyl), —NHC(O)N(C1-C4 alkyl)₂, —NH—C(═NH)NH₂, —SO₂NH₂—SO₂NH(C1-C3alkyl), —SO₂N(C1-C3 alkyl)₂, NHSO₂H, NHSO₂(C1-C4 alkyl) and aryl.Preferred substituents on aryl groups are as defined throughout thespecification.

Examples of suitable substituents on a substitutable ring nitrogen atomof an aryl group include C1-C4 alkyl, NH₂, C1-C4 alkylamino, C1-C4dialkylamino, —C(O)NH₂, —C(O)NH(C1-C4 alkyl), —C(O)(C1-C4 alkyl),—CO₂R**, —C(O)C(O)R**, —C(O)CH₃, —C(O)OH, —C(O)O—(C1-C4 alkyl),—SO₂NH₂—SO₂NH(C1-C3alkyl), —SO₂N(C1-C3alkyl)₂, NHSO₂H, NHSO₂(C1-C4alkyl), —C(═S)NH₂, —C(═S)NH(C1-C4 alkyl), —C(═S)N(C1-C4 alkyl)₂,—C(═NH)—N(H)₂, —C(═NH)—NH(C1-C4 alkyl) and —C(═NH)—N(C1-C4 alkyl)₂.

Substituted alkyl, aliphatic, non-aromatic carbocyclic or heterocyclicgroup as defined herein contain one or more substituents. Examples ofsuitable substituents for an alkyl group include those listed above fora substitutable carbon of an aryl and aliphatic and the following: ═O,═S, ═NNHR**, ═NN(R**)₂, ═NNHC(O)R**, ═NNHCO₂ (alkyl), ═NNHSO₂ (alkyl),═NR**, spiro cycloalkyl group or fused cycloalkyl group. R** in eachoccurrence, independently is —H or C1-C6 alkyl. Preferred substituentson alkyl aliphatic, non-aromatic carbocyclic or heterocyclic groupgroups are as defined throughout the specification.

In Vitro Testing and Use

The compounds (fluorochromes) and biocompatible fluorescent molecules ofthe present invention can be tested in vitro by one skilled in the artto assess its biological and performance characteristics. For instance,different types of cells grown in culture can be used to assess theirbiological and performance characteristics. Uptake, labeling, bindingtargeting or cellular localization of the fluorochromes andbiocompatible fluorescent molecules can be assessed using techniquesknown in the art such as spectroscopy methods, fluorescent microscopy,and flow cytometry. For example, the fluorochromes and biocompatiblefluorescent molecules of the present invention can be contacted with asample for a period of time and then washed to remove any free orunbound molecules. The sample can then be viewed using a fluorescentmicroscope equipped with appropriate filters matched to the opticalproperties of the fluorochromes and biocompatible fluorescent moleculesof the present invention. Fluorescent microscopy of cells in culture isalso a convenient means for determining whether uptake and bindingoccurs in one or more subcellular compartments. Tissues, tissue sectionsand other types of samples such as cytospin samples can also be used ina similar manner to assess the biological and performancecharacteristics of the molecules. Other fluorescent detection methodsincluding, but not limited to flow cytometry, immunoassays,hybridization assays, and microarray analysis can also be used.

Optical Imaging

The general principles of fluorescence, optical image acquisition, andimage processing can be applied in the practice of the invention. For areview of optical imaging techniques, see, e.g., Alfano et al., Ann. NYAcad. Sci. 820:248-270, 1997.

An imaging system useful in the practice of this invention typicallyincludes three basic components: (1) an appropriate light source forfluorochrome and biocompatible fluorescent molecule excitation, (2) ameans for separating or distinguishing emissions from light used for theexcitation, and (3) a detection system to detect the optical signalemitted.

In general, the optical detection system can be viewed as including aoptical gathering/image forming component and a optical detection/imagerecording component. Although the optical detection system can be asingle integrated device that incorporates both components, the opticalgathering/image forming component and light detection/image recordingcomponent will be discussed separately.

A particularly useful optical gathering/image forming component is anendoscope. Endoscopic devices and techniques which have been used for invivo optical imaging of numerous tissues and organs, includingperitoneum (Gahlen et al., J. Photochem. Photobiol. B 52:131-135, 1999),ovarian cancer (Major et al., Gynecol. Oncol. 66:122-132, 1997), colonand rectum (Mycek et al., Gastrointest. Endosc. 48:390-394, 1998; andStepp et al., Endoscopy 30:379-386, 1998), bile ducts (Izuishi et al.,Hepatogastroenterology 46:804-807, 1999), stomach (Abe et al., Endoscopy32:281-286, 2000), bladder (Kriegmair et al., Urol. Int. 63:27-31, 1999;and Riedl et al., J. Endourol. 13:755-759, 1999), lung (Hirsch et al.,Clin Cancer Res 7:5-220, 2001), brain (Ward, J. Laser Appl. 10:224-228,1998), esophagus, and head and neck regions can be employed in thepractice of the present invention.

Other types of optical gathering components useful in the invention arecatheter-based devices, including fiber optics devices. Such devices areparticularly suitable for intravascular imaging. See, e.g., Tearney etal., Science 276:2037-2039, 1997; and Circulation 94:3013, 1996.

Still other imaging technologies, including phased array technology(Boas et al., Proc. Natl. Acad. Sci. USA 91:4887-4891, 1994; Chance,Ann. NY Acad. Sci. 838:29-45, 1998), optical tomography (Cheng et al.,Optics Express 3:118-123, 1998; and Siegel et al., Optics Express4:287-298, 1999), intravital microscopy (Dellian et al., Br. J. Cancer82:1513-1518, 2000; Monsky et al, Cancer Res. 59:4129-4135, 1999; andFukumura et al., Cell 94:715-725, 1998), confocal imaging (Korlach etal., Proc. Natl. Acad. Sci. USA 96:8461-8466, 1999; Rajadhyaksha et al.,J. Invest. Dermatol. 104:946-952, 1995; and Gonzalez et al., J. Med.30:337-356, 1999) and fluorescence molecular tomography (FMT)(Nziachristos et al., Nature Medicine 8:757-760, 2002; U.S. Pat. No.6,615,063, PCT Application No. WO 03/102558, and PCT US/03/07579) can beemployed in the practice of the present invention, the IVIS® ImagingSystem (Xenogen, Alameda, Calif.), Maestro (CRI, Woburn, Mass.) theSoftScan® and the eXplore Optix™ (Advanced Research Technologies,Montreal, Canada) system can be employed in the practice of the presentinvention.

A suitable optical detection/image recording component, e.g., chargecoupled device (CCD) systems or photographic film, can be used in theinvention. The choice of optical detection/image recording will dependon factors including type of optical gathering/image forming componentbeing used. Selecting suitable components, assembling them into anoptical imaging system, and operating the system is within ordinaryskill in the art.

Diagnostic and Disease Applications and Methods

The methods of the invention can be used to determine a number ofindicia, including tracking the localization of the fluorochromes andbiocompatible fluorescent molecules in the subject over time orassessing changes or alterations in the metabolism and/or excretion ofthe molecules in the subject over time. The methods can also be used tofollow therapy for such diseases by imaging molecular events andbiological pathways modulated by such therapy, including but not limitedto determining efficacy, optimal timing, optimal dosing levels(including for individual patients or test subjects), and synergisticeffects of combinations of therapy.

The invention can be used to help a physician or surgeon to identify andcharacterize areas of disease, such as arthritis, cancers andspecifically colon polyps, or vulnerable plaque, to distinguish diseasedand normal tissue, such as detecting tumor margins that are difficult todetect using an ordinary operating microscope, e.g., in brain surgery,help dictate a therapeutic or surgical intervention, e.g., bydetermining whether a lesion is cancerous and should be removed ornon-cancerous and left alone, or in surgically staging a disease, e.g.,intraoperative lymph node staging, sentinel lymph node mapping, orassessing intraoperative bleeding.

The methods of the invention can also be used in the detection,characterization and/or determination of the localization of a disease,especially early disease, the severity of a disease or adisease-associated condition, the staging of a disease, and monitoringand guiding various therapeutic interventions, such as surgicalprocedures, and monitoring drug therapy, including cell based therapies.The methods of the invention can also be used in prognosis of a diseaseor disease condition. Examples of such disease or disease conditionsinclude inflammation (e.g., inflammation caused by arthritis, forexample, rheumatoid arthritis), cancer (e.g., colorectal, ovarian, lung,breast, prostate, cervical, skin, brain, gastrointestinal, mouth,esophageal, bone), cardiovascular disease (e.g., atherosclerosis andinflammatory conditions of blood vessels, ischemia, stroke, thrombosis),dermatologic disease (e.g., Kaposi's Sarcoma, psoriasis), ophthalmicdisease (e.g., macular degeneration, diabetic retinopathy), infectiousdisease (e.g., bacterial, viral, fungal and parasitic infections,including Acquired Immunodeficiency Syndrome), immunologic disease(e.g., an autoimmune disorder, lymphoma, multiple sclerosis, rheumatoidarthritis; diabetes mellitus), central nervous system disease (e.g., aneurodegenerative disease, such as Parkinson's disease or Alzheimer'sdisease), inherited diseases, metabolic diseases, environmental diseases(e.g., lead, mercury and radioactive poisoning, skin cancer), andbone-related disease (e.g., osteoporosis, primary and metastatic bonetumors, osteoarthritis). The methods of the invention can therefore beused, for example, to determine the presence of tumor cells andlocalization of tumor cells, the presence and localization ofinflammation, including the presence of activated macrophages, forinstance in atherosclerosis or arthritis, the presence and localizationof vascular disease including areas at risk for acute occlusion (i.e.,vulnerable plaques) in coronary and peripheral arteries, regions ofexpanding aneurysms, unstable plaque in carotid arteries, and ischemicareas. The methods and compounds (compositions) of the invention canalso be used in identification and evaluation of apoptosis, necrosis,hypoxia and angiogenesis.

Optical imaging modalities and measurement techniques include, but arenot limited to, fluorescence imaging; endoscopy; fluorescence endoscopy;optical coherence tomography; transmittance imaging; time resolvedtransmittance imaging; confocal imaging; nonlinear microscopy;photoacoustic imaging; acousto-optical imaging; spectroscopy;reflectance spectroscopy; intravital imaging; two photon imaging;interferometry; coherence interferometry; diffuse optical tomography andfluorescence molecular tomography, and measurement of light scattering,absorption, polarisation, luminescence, fluorescence lifetime, quantumyield, and quenching.

The compounds (compositions) and methods of the present invention can beused in combination with other imaging compositions and methods. Forexample, the methods of the present invention can be used in combinationwith other traditional imaging modalities such as X-ray, computedtomography (CT), positron emission tomography (PET), single photoncomputerized tomography (SPECT), and magnetic resonance imaging (MRI).For instance, the compounds (compositions) and methods of the presentinvention can be used in combination with CT and MR imaging to obtainboth anatomical and biological information simultaneously, for example,by co-registration of a tomographic image with an image generated byanother imaging modality. In particular, the combination with MRI or CTis preferable, given the high spatial resolution of these imagingtechniques. The compounds (compositions) and methods of the presentinvention can also be used in combination with X-ray, CT, PET, SPECT andMR contrast agents or the fluorescent silicon nanoparticle imagingprobes of the present invention may also contain components, such asiodine, gadolinium atoms and radioactive isotopes, which can be detectedusing CT, PET, SPECT, and MR imaging modalities in combination withoptical imaging.

Kits

The compounds (compositions) described herein can be packaged as a kit,which may optionally include instructions for using the fluorochromes orbiocompatible fluorescent molecules in various exemplary applications.Non-limiting examples include kits that contain, e.g., the compounds(compositions) in a powder or lyophilized form, and instructions forusing, including reconstituting, dosage information, and storageinformation for in vivo and/or in vitro applications. Kits mayoptionally contain containers of the compounds (compositions) in aliquid form ready for use, or requiring further mixing with solutionsfor administration. For in vivo applications, the kit may contain thecompounds (compositions) in a dosage and form suitable for a particularapplication, e.g. a liquid in a vial, a topical creams, etc.

The kit can include optional components that aid in the administrationof the unit dose to subjects, such as vials for reconstituting powderforms, syringes for injection, customized IV delivery systems, inhalers,etc. The kits may be supplied in either a container which is providedwith a seal which is suitable for single or multiple puncturing with ahypodermic needle (e.g. a crimped-on septum seal closure) whilemaintaining sterile integrity. Such containers may contain single ormultiple subject doses. Additionally, the unit dose kit can containcustomized components that aid in the detection of the compounds(compositions) in vivo or in vitro, e,g., specialized endoscopes, lightfilters. The kits may also contain instructions for preparation andadministration of the compounds (compositions). The kit may bemanufactured as a single use unit dose for one subject, multiple usesfor a particular subject; or the kit may contain multiple doses suitablefor administration to multiple subjects (“bulk packaging”). The kitcomponents may be assembled in cartons, blister packs, bottles, tubes,and the like.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES

The following non limiting examples demonstrate the synthesis ofnicotinic acid and picolinic acid derived near infrared fluorophores.Representative materials and methods that may be used in preparing thecompounds of the invention are described further below. All chemicalsand solvents (reagent grade) were used as supplied from the vendorscited without further purification.

The analytical and preparative HPLC methods generally utilized are:

-   A Column: Agilent Zorbax 80 Å, Extend C18, 4.6×250 mm (5 μm). Mobile    Phase: Acetonitrile and 25 mM triethylammonium acetate.-   B Column: Varian Dynamax, 100 Å, C18, 41.4×250 mm. Mobile Phase:    Acetonitrile and 25 mM triethylammonium acetate.-   C Column: Phenomenex Jupiter, 300 Å, C18 Mobile Phase: Acetonitrile    and 25 mM triethylammonium acetate.

Example 1 Synthesis of Example 1

Part A. 6-hydrazino-1,3-naphthalene disulfonate (I)

6-Amino-1,3-naphthalene disulfonate disodium salt (10 g, 29 mmol) (TCI)was dissolved in 30 mL of water and added to 50 mL of water and 15 mL ofconcentrated hydrochloric acid. The slurry was cooled to <0° C. in anice/acetone bath and sodium nitrite (2.2 g, 32 mmol, Aldrich) was addedin 40 mL of cold water drop-wise over 10 minutes. Stannous chloride (11g, 58 mmol) (Aldrich) was dissolved in 30 mL water and 6 mL concentratedhydrochloric acid, cooled to 0° C. and added to the reaction mixtureover 10 minutes. The resulting solution was stirred and allowed to warmto room temperature over 3 hours resulting in a clear, brown solution.The solution was reduced in volume by rotary evaporation and the productprecipitated by the addition of isopropanol. Product (I) was filtered,washed with isopropanol and dried in vacuum.

Part B. 2,3,3-trimethylbenzindole-5,7-disulfonate (II)

6-Hydrazino-1,3-naphthalene disulfonated (10 g, 25 mmol), isopropylmethyl ketone (12 g, 140 mmol, Aldrich) and potassium acetate (6 g, 61mmol) were combined in 75 mL glacial acetic acid and heated to 145° C.for 22 hours. The solution was cooled and the acetic acid was removed byrotary evaporation. The residue was dissolved in methanol and filtered.The product (II) was then precipitated from the methanol filtrate withisopropanol and filtered, washed with isopropanol and ether and dried invacuum.

Part C.2,3,3-trimethyl-1-(3-sulfonatopropyl)benzindolinium-5,7-disulfonate(III)

2,3,3-Trimethylbenzindole-6,8-disulfonate (2.2 g, 5 mmol) was dissolvedin 50 mL of dry DMF resulting in a clear orange solution.1,3-propanesultone (2.8 g, 23 mmol, Aldrich) was added and the solutionwas heated to 145° C. in a sealed tube for 15 hours, turning dark purplein color. The solution was cooled and poured into 150 mL 2-propanol. Themixture was centrifuged and the solvent decanted off. The solid productwas washed on a filter with three 50 mL portions of 2-propanol followedby 50 mL of ether and dried in vacuum, resulting in 2.5 g of a darkpurple solid (90%). MALDI-TOF-MS m/e 492.05 [M]+ calculated forC₁₈H₂₂NO₉S₃ ⁺, found 492.05.

Part D: Preparation of Compound IV

A 100 mL round bottom flask, fitted with a reflux condenser was chargedwith 2,3,3-trimethyl-1-(3-sulfonatopropyl)benzindolinium-5,7-disulfonate(565 mg, 1 mmol), 2-(3-hydroxycarbonyl-6-pyridyl)-malondialdehyde (98mg, 0.5 mmol), and sodium acetate (585 mg, 7.1 mmol). Acetic anhydride(10 mL) and acetic acid (10 mL) were added to the flask, and the mixturewas heated to reflux for 8 h, cooled down to room temp and 25 mL ofethyl acetate was added. The dark blue dye precipitate was collected byfiltration, which was dissolved in 20 mL of water and purified bypreparative reversed phase HPLC to afford the compound IV as powder.Yield, 285 mg, 50%. MALDI MS: Expected, 1139.09; Found, 1139.6732

Part E: Preparation of Example 1

Compound IV (11.4 mg, 0.01 mmol) was mixed with disuccinimidyldicarbonate (DSC, 5 mg, 0.02 mmol)) and N,N-dimethylaminopyridine (DMAP,2 mg, 0.016 mmol)) in 500 μL of dry DMF, and heated to 70° C. After 30min cold ethyl acetate (500 μL) was added to the reaction mixture toprecipitate the product out, which was filtered, dried under vacuum andstored at 4° C.

Example 2 Synthesis of Example 2

Part A: Preparation of Compound V

Compound V was prepared from potassium,2,3,3-trimethyl-1-ethyl-benzindolinium-5,7-disulfonate (184 mg, 0.423mmol), 2-(3-hydroxycarbonyl-6-pyridyl)-malondialdehyde (40 mg, 0.21mmol), and sodium acetate (300 mg, 3.66 mmol) mixed in 6 mL of aceticanhydride and 6 mL of acetic acid. After heating at 125° C. for 16hours, the product was precipitated from ethyl acetate and purified byreversed phase HPLC. The product in a powder form was obtained afterspeed vacuum drying of the HPLC fractions. MALDI. m/z calculated (found)for M⁺: 952.05 (952.16). Yield, 140 mg, 70%.

Part B: Preparation of Example 2

One equivalent of Compound V was mixed with two equivalents ofdisuccinimidyl dicarbonate (DSC) and one equivalent ofN,N-dimethylaminopyridine (DMAP) in 0.5 mL of DMF, and heated to 70° C.The reaction was complete within 30 min; the desired product wasprecipitated from the DMF solution by the addition of ethyl acetate,filtered, dried under vacuum and stored at 4° C.

Example 3 Synthesis of Example 3

Part A: Preparation of Compound VI

Compound VI was prepared from2,3,3-Trimethyl-1-(3-sulfonatopropyl)-3H-benzindolinium-5,7-disulfonate(101 mg, 0.18 mmol), 2-(2-hydroxycarbonyl-6-pyridyl)-malonaldialdehyde(17.4 mg, 0.09 mmol), and sodium acetate (88 mg, 1.07 mmol) mixed in 6mL of acetic anhydride and 6 mL of acetic acid. After heating at 125° C.for 4 hours, the product was precipitated from ethyl acetate andpurified by reversed phase HPLC. The product was obtained in a powderform after speed vacuum drying of the HPLC fractions. MALDI. m/zcalculated (found) for (M+1)⁺: 1140.33 (1140.13). Yield, 67 mg, 65%.

Part B: Preparation of Example 3

One equivalent of compound VI was mixed with two equivalents ofdisuccinimidyl dicarbonate (DSC) and one equivalent ofN,N-dimethylaminopyridine (DMAP) in 0.5 mL of DMF, and heated to 70° C.The progress of the reaction was monitored by HPLC. The reaction wascomplete within 30 min, following which the desired product wasprecipitated from the DMF solution by the addition of ethyl acetate,filtered, dried under vacuum and stored at 4° C.

Example 4 Synthesis of Example 4

Example 4 is prepared from2,3,3-Trimethyl-1-(ethyl)-3H-benzindolinium-5,7-disulfonate (0.18 mmol),2-(2-hydroxycarbonyl-6-pyridyl)-malonaldialdehyde (0.09 mmol), andsodium acetate (88 mg, 1.07 mmol) mixed in 6 mL of acetic anhydride and6 mL of acetic acid. After heating at 125° C. for 4 hours, the productis precipitated from ethyl acetate, purified by reversed phase HPLC andobtained in a powder form after speed vacuum drying of the HPLCfractions.

Example 5 Synthesis of Example 5

2,3,3-Trimethyl-1-(3-sulfonatopropyl)-indolinium-5-sulfonate (115 mg,0.28 mmol), 2-(3-hydroxycarbonyl-6-pyridyl)-malonaldialdehyde (27 mg,0.14 mmol), and sodium acetate (80 mg, 1.0 mmol) were mixed in 5 mL ofacetic anhydride and 5 mL of acetic acid. After heating at 125° C. for 2hours, reaction mixture was cooled down and the product was precipitatedfrom ethyl acetate, followed by purification by reversed phase HPLC. Theproduct was obtained in a powder form after speed vacuum drying of theHPLC fractions. Yield, 87 mg, 70%. Abs. max (water): 635 nm; Em. Max(water): 653 nm.

Example 6 Synthesis of Example 6

2-Methyl-1(3-sulfonatopropyl)-benzothiazolium inner salt (150 mg, 0.55mmol) and 2-(3-hydroxycarbonyl-6-pyridyl)-malonaldialdehyde (52 mg, 0.27mmol) were added to a mixture of 2-methoxy ethanol and toluene (2:1,v/v) and heated to reflux at 150° C. attached to a Dean-Stark condenser.After 4 hours, heating was stopped, cooled down and solvents wereremoved by rotary evaporation. The concentrated blue dye was purifiedusing preparative HPLC. The product was obtained in a powder form afterdrying of the HPLC fractions on speed vacuum. Yield, 95 mg, 50%. Abs.max (water): 643 nm; Em. Max (water): 663 nm.

Example 7 Synthesis of Example 7

10 mg of the NHSE of Compound IV was dissolved in 100 uL dry DMF, towhich was added a solution of 3-azidopropylamine (5 mg) in 20 uL dryDMSO, and the mixture was rotated at room temperature for one hour. 1 mLether was added to the reaction mixture, and centrifuged for 10 minutes.The supernatant solution was discarded, and the residue was dried onspeed vac for 5 minutes, redissolved in water and purified on a RPC18semi-prep column. The fraction corresponding to the product (Example 7)was collected, and dried on speedvac which yielded 7.2 mg (60%). It wascharacterized by MALDI. Calculated: 1222.34. Found: 1222.54

Example 8 Synthesis of Example 8

10 mg of the NHSE of Compound IV was dissolved in 100 uL dry DMF, towhich was added a solution of 3-propargylamine (5 mg) in 10 uL dry DMSO,and the mixture was rotated at room temperature for one hour. 1 mL etherwas added to the reaction mixture, and centrifuged for 10 minutes. Thesupernatant solution was discarded, and the residue was dried on speedvac for 5 minutes, redissolved in water and purified on a RPC18semi-prep column. The fraction corresponding to the product (Example 8)was collected, and dried on speedvac. Yield: 6 mg (52%). It wascharacterized by MALDI. Calculated: 1177.32. Found: 1177.21

Example 9 Synthesis of Example 9

17 mg of the NHSE of Compound IV was dissolved in 250 uL dry DMF, towhich was added a solution of 2-(2-aminoethyl-dithio)pyridinehydrochloride (11 mg) in 10 uL dry DMSO and 4 uL of triethylamine, andthe mixture was rotated at room temperature for an overnight. 1 mL ethylacetate was added to the reaction mixture, and centrifuged for 10minutes. The supernatant solution was discarded, and the residue wasdried on speed vac for 5 minutes, redissolved in water and purified on aRPC18 semi-prep column. The fraction corresponding to the product(Example 9) was collected, and dried on speedvac. Yield: 10 mg (65%). Itwas characterized by MALDI. Calculated: 1308.52. Found: 1308.54

Example 10 Cell Labeling

Mouse splenocytes were prepared as a single cell suspension, and the Tcell subpopulation within the splenocyte preparation were enriched bypassage over a column that removes B cells and macrophages (R& D kit,Mouse T-cell enrichment columns, MTCC500). T cells were centrifuged togenerate a cell pellet of 10⁷ cells. The supernatant was removed fromthe cell pellet, and a solution of 10 mg/ml of compound of Example 1 in100 uL was added. Cells were incubated at room temperature for 5minutes, followed by 2 rounds of centrifugation and resuspension inphysiologic buffer to wash away unbound Example 1. Cells were assessedby fluorescence microscopy.

Example 11 Cell Labeling and in Vivo Imaging

Mouse 4T1 breast adenocarcinoma cells were centrifuged to generate acell pellet of 10⁷ cells. The supernatant was removed from the cellpellet, and a solution of 10 mg/ml Example 1 in 100 uL was added. Cellswere incubated at room temperature for 5 minutes, followed by 2 roundsof centrifugation and resuspension in physiologic buffer to wash awayunbound Example 1. Cells were assessed by fluorescence microscopy. Cellswere injected intravenously into mice at 5×10⁵ cells per mouse, and livemice were imaged by fluorescent molecular tomography immediately afterinjection and 24 hours after injection. As 4T1 cells primarilymetastasize to the lungs, lung fluorescence is quantified.

Example 12

A solution of Example 1 was chemically linked to an amine presentingsurface of nanoparticles to yield a biocompatible fluorescent moleculefor in vivo optical imaging. The tumor cell line HT-29 (human coloncarcinoma/HTB-38) was obtained from ATCC (Manassas, Va.). HT-29 cellswere grown in McCoy's supplemented with 10% FBS at 37° C. in ahumidified atmosphere containing 5% CO₂. Exponentially growing cellswere trypsinized and re-suspended in Hank's Balanced Salt Solution at aconcentration of 3×10⁷ cells/ml. Female NU/NU mice 6-8 weeks old(Charles River Laboratory, Wilmington, Mass.) were injectedsubcutaneously with 3×10⁶ HT-29 cells bilaterally in the first mammaryfat pads. One week later, when tumors were approximately 30 mm³, micewere injected intravenously with the fluorescent molecule, (in 150 μl of1×PBS) and imaged after 24 hrs on a fluorescence reflectance system(FRI, Kodak 2000MM) system. Results are shown in FIG. 1

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-27. (canceled)
 28. A biocompatible fluorescent molecule comprising oneor more biomolecules chemically linked to a compound represented byformula (2):

or a salt thereof, wherein: X is independently selected from the groupconsisting of C(CH₂Y₁)(CH₂Y₂), O, S, and Se; Y₁ and Y₂ are independentlyselected from the group consisting of H, C₁-C₂₀ aliphatic group and aC₁-C₂₀ aliphatic group substituted with —OR*, N(R*)₂ or —SR*; Wrepresents a benzo-condensed, a naphtho-condensed or a pyrido-condensedring; R₁ is selected from the group consisting of H, (CH₂)_(x)CH₃,(CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein x is an integer selected from0 to 6 and n is an integer selected from 2 to 6; R₄ is selected from thegroup consisting of H, (CH₂)_(x)CH₃, (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H,wherein x is an integer selected from 0 to 6 and n is an integerselected from 2 to 6; R₂ and R₃ are independently selected from thegroup consisting of H, carboxylate, carboxylic acid, carboxylic ester,amine, amide, sulfonamide, hydroxyl, alkoxyl, a sulphonic acid moietyand a sulphonate moiety; Q is selected from a group consisting of aheteroaryl ring substituted with a carboxyl group or 6-memberedheteroaryl ring substituted with a carbonyl group.
 29. The biocompatiblefluorescent molecule of claim 28, wherein the compound has an absorptionand emission maxima between about 500 nm and about 900 nm.
 30. Thebiocompatible fluorescent molecule of claim 29, wherein the compound hasan absorption and emission maxima between about 600 nm and 800 nm. 31.The biocompatible fluorescent molecule of claim 28, wherein thebiocompatible fluorescent molecule is activated after targetinteraction.
 32. The biocompatible fluorescent molecule of claim 28,wherein the biocompatible fluorescent molecule has a high bindingaffinity to a target.
 33. A method of in vivo optical imaging, themethod comprising: (a) administering to a subject a biocompatiblefluorescent molecule comprising one or more biomolecules chemicallylinked to a compound represented by formula (2):

or a salt thereof, wherein: X is independently selected from the groupconsisting of C(CH₂Y₁)(CH₂Y₂), O, S, and Se; Y₁ and Y₂ are independentlyselected from the group consisting of H, C₁-C₂₀ aliphatic group and aC₁-C₂₀ aliphatic group substituted with —OR*, N(R*)₂ or —SR*; Wrepresents a benzo-condensed, a naphtho-condensed or a pyrido-condensedring; R₁ is selected from the group consisting of H, (CH₂)_(x)CH₃,(CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein x is an integer selected from0 to 6 and n is an integer selected from 2 to 6; R₄ is selected from thegroup consisting of H, (CH₂)_(x)CH₃, (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H,wherein x is an integer selected from 0 to 6 and n is an integerselected from 2 to 6; R₂ and R₃ are independently selected from thegroup consisting of H, carboxylate, carboxylic acid, carboxylic ester,amine, amide, sulfonamide, hydroxyl, alkoxyl, a sulphonic acid moietyand a sulphonate moiety; Q is selected from a group consisting of aheteroaryl ring substituted with a carboxyl group or 6-memberedheteroaryl ring substituted with a carbonyl group; (b) allowing time forthe biocompatible fluorescent molecule to distribute within the subjector to contact or interact with a biological target; (c) illuminating thesubject with light of a wavelength absorbable by the biocompatiblefluorescent molecule; and (d) detecting the optical signal emitted bythe biocompatible fluorescent molecule.
 34. The method of claim 33,wherein the signal emitted by the biocompatible fluorescent molecule isused to construct an image.
 35. The method of claim 34, wherein steps(a)-(d) are repeated at predetermined intervals thereby allowing forevaluation of the emitted signals of the biocompatible fluorescentmolecule in the subject over time.
 36. The method of claim 35, whereinthe subject is an animal or human.
 37. The method of claim 36, whereinin step (a) two or more fluorescent molecule whose signal properties aredistinguishable are administered to a subject.
 38. The method of claim37, wherein the illuminating and detecting steps are performed using anendoscope, catheter, tomographic system, hand-held optical imagingsystem, surgical goggles, or intra-operative microscope.
 39. The methodof claim 38, wherein the presence, absence, or level of signal emittedby the fluorescent molecule is indicative of a disease state.
 40. Themethod of claim 39, wherein the method is used to detect and monitordisease.
 41. The method of claim 40, wherein the disease is selectedfrom the group consisting of cancer, cardiovascular diseases,neurodegenerative diseases, immunologic diseases, autoimmune diseases,respiratory diseases, metabolic diseases, inherited diseases, infectiousdiseases, bone diseases, and environmental diseases.
 42. The method ofclaim 41, wherein prior to step (a) the fluorescent molecule of claim 1is mixed with cells to label the cells and the resulting labeled cellsare administered to the subject in step (a).
 43. The method of claim 42,wherein the signal emitted by the fluorescent molecule is used formonitoring trafficking and localization of cells or evaluation a celltherapy.
 44. A biocompatible fluorescent molecule comprising abiomolecule chemically linked to a compound represented by formula (2):

or a salt thereof, wherein: X is independently selected from the groupconsisting of C(CH₂Y₁)(CH₂Y₂), O, S, and Se; Y₁ and Y₂ are independentlyselected from the group consisting of H, C₁-C₂₀ aliphatic group and aC₁-C₂₀ aliphatic group substituted with —OR*, N(R*)₂ or —SR*; Wrepresents a benzo-condensed, a naphtho-condensed or a pyrido-condensedring; R₁ is selected from the group consisting of H, (CH₂)_(x)CH₃,(CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein x is an integer selected from0 to 6 and n is an integer selected from 2 to 6; R₄ is selected from thegroup consisting of H, (CH₂)_(x)CH₃, (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H,wherein x is an integer selected from 0 to 6 and n is an integerselected from 2 to 6; R₂ and R₃ are independently selected from thegroup consisting of H, carboxylate, carboxylic acid, carboxylic ester,amine, amide, sulfonamide, hydroxyl, alkoxyl, a sulphonic acid moietyand a sulphonate moiety; Q is selected from a group consisting of aheteroaryl ring substituted with carboxyl group or 6-membered heteroarylring substituted with a carbonyl group; wherein the biomolecule is alabeled cell.